gift fromthierrygalli  (Addgene inc)

Journal: Nature Communications

Article Title: Spatiotemporal proteomics reveals the biosynthetic lysosomal membrane protein interactome in neurons

doi: 10.1038/s41467-024-55052-w

Figure Lengend Snippet: a Heatmap in which the Log 2 Fold Change across time points of axonal synaptic proteins and members of the SNARE complex is plotted (two-sided t-test, no multiple comparison test was used). b Live neuron expressing RUSH-LAMP2A-mNG, RUSH-SYT1-Halo and scramble, imaged during 1 h of biotin addition. Still images show part of the Golgi and a budding event. Intensity profile graph in the bottom. c Neurons expressing RUSH-LAMP2A-mNG and RUSH-SYT1-Halo at 1 and 4 h post-release. Kymographs from live cell imaging along the axon every 1 s for 180 s are shown. Colocalized anterograde (blue), retrograde (orange) or stationary (grey) trajectories were traced on the right. d Quantification of the number of trajectories for 1 and 4 h. n = 15 and 19 neurons; each from 3 independent experiments ( N = 3; **** p < 0.0001, * p = 0.0433, ns p = 0.6092) e Confocal images of neurons expressing RUSH-LAMP1-V5 and EGFP-VAMP4, 1 h after release. Blue and orange boxes indicate magnified areas shown on the right, with corresponding intensity profile graph. f , g Confocal images of neurons expressing RUSH-LAMP1-V5 ( f ) or RUSH-SYT1-mNG ( g ) plus shRNA against VAMP4, or scramble. Quantifications of the number of RUSH-LAMP1 ( n = 19 and 27 neurons; each from 4 independent experiments N = 4, **** p < 0.0001) and SYT1 ( n = 27 and 22 cells; each from 3 independent experiments N = 3, **** p < 0.0001) positive compartments are shown on the right. h Still images from the soma of neuron expressing RUSH-LAMP2a-mNG, RUSH-SYT1-Halo and shRNA against VAMP4; control scramble in ( b ). Images show a Golgi budding event after 1 h release. Corresponding intensity profile graph on the right. i Neurons expressing RUSH-LAMP2A-mNG and shRNA against VAMP4 or scramble after 1 h release and immunostained for LAMTOR4 with respective intensity profile graphs are shown. j Neurons in ( h ) were labeled for SirLyso and imaged live after 1 h release. Still images from time points indicated in images and respective intensity profile graphs are shown. k Temporal intensity profile graph for RUSH-SYT1 and SirLyso from image in ( j ). Scale bars, 2 µm in ( b ), ( h – j ), 5 µm in ( f ), ( g ), and 10 µm in ( e ). Data are presented as mean values ± SD, plus individual points. Mann-Whitney test was used in ( d ), ( f ), and ( g ). See also Supplementary Figs. and . Representative images in b , e , and i were repeated in at least 3 independent experiments. Source data are provided as a Source Data file.

Article Snippet: The following vectors were used: FUGW was a gift from David Baltimore (Addgene plasmid # 14883) , pLKO.1 puro was a gift from Bob Weinberg (Addgene plasmid # 8453) , psPAX2 and pMD2.G were gifts from Didier Trono (Addgene plasmids # 12260 and # 12259) pmScarlet-i_C1 was a gift from Dorus Gadella (Addgene plasmid # 85044) , H2B-mNeonGreen-IRESpuro2 was a gift from Daniel Gerlich (Addgene plasmid # 183745) , LAMP1-RFP was a gift from Walther Mothes (Addgene plasmid # 1817) , pEGFP-VAMP4 was a gift from Thierry Galli (Addgene plasmid # 42313) , Str-KDEL_SBP-EGFP-E-cadherin was a gift from Franck Perez (Addgene plasmid # 65286) , mito-V5-APEX2 was a gift from Alice Ting (Addgene plasmid # 72480) , pAAV hSyn GFP-FXR1 was a gift from Martin Beaulieu (Addgene plasmid # 112732) , LAMP1-GFP was a gift from Dr. Juan Bonifacino, GFP-RAB6A, GFP-RAB7A and GFP-RAB11A were gifts from Casper Hoogenraad , pAAV ORANGE Gria1-HaloTag was a gift from Harold MacGillavry, PB-Ef1a-PCP-Halo (Addgene plasmid # 198337) and PB-Ef1a-β-actin-UTR-PP7 mRNA were gifts from Michael Ward.

Techniques: Comparison, Expressing, Live Cell Imaging, shRNA, Control, Labeling, MANN-WHITNEY

Journal: The Journal of cell biology

Article Title: Vesicle-associated membrane protein 2 is a cargo-selective v-SNARE for a subset of GPCRs.

doi: 10.1083/jcb.202207070

Figure Lengend Snippet: Figure 2. VAMP2 is detected preferentially in fusion events (puffs) delivering MOR to the surface. (A) Schematic of a VAMP2+ puff in SpH-cargo and VAMP2-pHuji co-expressing cells. When a vesicle carrying both SpH-cargo and VAMP2-pHuji fuses to the plasma membrane, the fluorescence intensity increases in both the SpH channel and the pHuji channel. (B) Montage of a SpH-MOR puff colocalizing with VAMP2-pHuji (SpH-MOR in green and VAMP2-pHuji in magenta) from SpH-MOR and VAMP2-pHuji co-expressing cells 5 min after DAMGO treatment at 37°C. Scale bar = 2 µm. (C) Representative time-course traces of mean SpH-MOR (cyan) and the corresponding mean VAMP2-pHuji fluorescence (magenta) from a VAMP2+ puff of the SpH-MOR expressing cells. (D) Representative time-course traces of mean SpH-B2AR (red) and the corresponding mean VAMP2-pHuji fluorescence (magenta) from a VAMP2-negative puff of the SpH-B2AR expressing cells. (E) Quantification of VAMP enrichment in the population of puffs for each SpH-cargo. VAMP2 or VAMP4 enrichment of individual puff event was calculated as the fold change of VAMP-pHuji fluorescence at the peak of the event over baseline SD, as described in the text (median ± 95% confidence interval [CI]; TfR-SpH: 2 cells, puff = 85; SpH-B2AR: 3 cells, puff = 85; SpH-MOR-VAMP2: 3 cells, puff = 59; SpH-MOR-VAMP4: 4 cells, puff = 44; all conditions from two independent experiments). SpH-MOR puffs showed significantly more enrichment of VAMP2 compared with other cargos, but showed no VAMP4 enrichment (one-way ANOVA, post-hoc Kruskal–Wallis test: TfR vs. B2AR: P > 0.9999; TfR vs. MOR: P < 0.0001; B2AR vs. MOR: P < 0.0001; MOR- VAMP2 vs. MOR-VAMP4: P < 0.0001). (F) The fraction of VAMP2+ puffs (% population) as defined by different folds of VAMP2 enrichment over baseline SD (cutoff) shows a consistent enrichment of VAMP2 preferentially in MOR puffs across all cutoffs. (G) Kernel density estimation of the pooled population of all puffs to estimate subclasses. The best fit predicted three subclasses based on VAMP2 enrichment. Three Gaussian mixture models of the actual distribution (solid line) and predicted subclasses (dashed lines) are shown. (H) Fraction of puffs in each predicted subclass shows distinct population composition of different SpH-cargos and a preferential enrichment of VAMP2+ subclasses in SpH-MOR puffs.

Article Snippet: VAMP4-pHuji plasmid expressing human VAMP4 was cloned by first replacing VAMP2 sequence of VAMP2-pHuji with a VAMP4 sequence from EGFP-VAMP4 (a gift from Thierry Galli, Université Paris Cité, Paris, France; #42313; Addgene) using XhoI and BamHI sites, and then the stop codon and original linker were replaced by VAMP2-pHuji’s linker.

Techniques: Expressing, Clinical Proteomics, Membrane, Fluorescence

Journal: The Journal of cell biology

Article Title: Vesicle-associated membrane protein 2 is a cargo-selective v-SNARE for a subset of GPCRs.

doi: 10.1083/jcb.202207070

Figure Lengend Snippet: Figure 3. VAMP2 depletion inhibits the recycling of SpH-MOR but not that of SpH-B2AR and TfR-SpH. (A) Representative confocal image of PC12 cells expressing shScramble-tagBFP (blue) immunostained for endogenous VAMP2 (magenta) after 48 h of Dox treatment. (B) Representative confocal image of PC12 cells expressing shVAMP2-tagBFP (blue) immunostained for endogenous VAMP2 (magenta) after 48 h of Dox treatment, showing depletion of VAMP2. Scale bar = 20 µm. (C) Scatter plots of normalized IDoT of VAMP2 immunostaining in shScramble-tagBFP (shScramble) or shVAMP2-tagBFP (shVAMP2) expressing cells (median ± 95% CI, n = 45 or 46 cells, respectively, from two independent experiments), showing depletion of VAMP2 in the latter (P < 0.0001, unpaired two-tailed t test). (D) The number of TfR-SpH puffs per cell per minute (/cell • min) in cells expressing either TfR-SpH alone (Dox alone) or in cells co- expressing shScramble or shVAMP2, treated with 48 h of Dox, show no significant difference (Dox alone: n = 11; shScramble: n = 10; shVAMP2: n = 10, from two independent experiments; one-way ANOVA, post-hoc Tukey test). (E) A similar comparison shows no difference in the numbers of SpH-B2AR puffs across the three conditions (Dox alone: n = 27; shScramble + Dox: n = 24; shVamp2 + Dox: n = 36, from three independent experiments, one-way ANOVA, post-hoc Tukey test). (F) A similar comparison shows significant reduction in the number of SpH-MOR puffs in cells co-expressing shVAMP2 compared with the other two conditions (Dox alone: n = 27; shScramble: n = 23; shVAMP2: n = 28, from three independent experiments, one-way ANOVA, post-hoc Tukey test). (G) A similar comparison in cells co-expressing SpH-MOR and shScramble or VAMP4 shRNA (shVAMP4), shows no difference in the numbers of SpH-MOR puffs (shScramble, n = 15; shVAMP2, n = 13, from one experiment, unpaired two-tailed t test, checked by power analysis). Cells from E–G were treated with agonists for 5 min at 37°C before imaging. Filled dots in red from E–G represented outliers identified by the Tukey plot that were included in the statistical analysis. “+” in the Tukey plots from D–G represented mean. (H) Schematic of the treatment and labeling conditions for the flow cytometry assay. Cells were labeled with M1-Alexa-647 (M1-647) antibody at the endpoint of treatment conditions to measure surface MOR levels: at baseline (NT), after DAMGO for 20 min at 37°C (T), or after washing out DAMGO and incubating in Naltrexone for 20 min at 37°C (WO). (I) Representative histograms of PC12 cells co-expressing FLAG-MOR and shVAMP2, either without (shVAMP2 −Dox) or with (shVAMP2 +Dox) pretreatment of Dox for 48 h. (J) Quantification of mean (left panel) and geometric mean (geomean, right panel) of surface MOR levels normalized to unlabeled baseline (±SEM, three samples each condition from one experiment).

Article Snippet: VAMP4-pHuji plasmid expressing human VAMP4 was cloned by first replacing VAMP2 sequence of VAMP2-pHuji with a VAMP4 sequence from EGFP-VAMP4 (a gift from Thierry Galli, Université Paris Cité, Paris, France; #42313; Addgene) using XhoI and BamHI sites, and then the stop codon and original linker were replaced by VAMP2-pHuji’s linker.

Techniques: Expressing, Immunostaining, Two Tailed Test, Comparison, shRNA, Imaging, Labeling, Flow Cytometry